The Molecular Biology of Bones and Teeth Unit is directed by Dr. Marian F. Young, and during this fiscal year included Drs. Xiao-Dong Chen, Sunil Wadhwa and Yanming Bi, with the technical assistance of Ms. Tina Kilts. The goal of this unit is to study the function of matrix proteins made by cells in skeletal tissues. Biglycan (bgn) is an extracellular protein that is highly expressed in the matrices of bones and teeth. To understand what role it plays in skeletal tissues, mice were created that were unable to make the protein (knockout/KO). These mice suffer from age dependent osteopenia with decreased bone mass and bone strength compared to wild-type (wt) mice. The goal of our current studies was to investigate the effects of physiological stress on the bones of the mutant mice. This was done to determine what function biglycan might play in regulating bone formation (osteogenesis), resorption (taking away bone) and turnover (coupling of the two processes) in live animals (in vivo). To accomplish this goal several different experimental approaches were used. First, the femora from 2 month-old normal and mutant mice were flushed with PBS to induce rapid and reproducible osteogenesis. Analysis of bone tissue by X-ray (2 dimensions) and pQCT (3 dimensions) showed that bgn deficient mice had significantly less accumulation of bone compared to normal mice proving that bgn is important for the osteogenesis in vivo. To test whether bgn could be involved in the effects of estrogen depletion on induced bone formation, we analyzed the bones from normal and bgn deficient mice after ovariectomy (OVX). Bgn deficient mice were resistant to OVX-induced trabecular bone loss where the normal mice responded as expected and displayed increased bone turnover. Analysis of the serum of normal and mutant mice showed increased levels of osteoprotogerin (OPG) and decreased levels of RANKL suggesting that altered production of these cytokines is one of the molecular bases for bgn's role in this process. Taken together, these data support the concept that bgn has dual roles in bone where it modulates both formation and resorption, ultimately influencing the bone turnover process. We are currently confirming this theory by subjecting mice to mechanical stimulation using forced treadmill running (to induce bone formation) and to induced osteolysis using titanium particles layered onto calvarial skull bones (to induce bone resporption). Now that we have shown that bgn is important for regulating bone tissue function, we wanted to determine the cell and molecular basis for its control. Previously we showed that bgn was important for TGF-beta induction of osteogenic bone marrow stromal cells. TGF-beta is part of a superfamily that includes the morphogenic proteins called BMPs. Using bone cells isolated from bgn deficient mice, we showed that the absence of bgn caused less BMP-4 binding, which reduced the sensitivity of osteoblasts to BMP-4 stimulation. The loss of sensitivity resulted in reduced expression of the osteogenic master gene, cbfa1, which ultimately led to a defect in the differentiation of osteoblasts. The response of bgn deficient osteoblasts to BMP-4 was completely rescued by reintroduction of bgn by viral transfection. Based on these observations, we theorize that bgn can modulate BMP-induced bone cell differentiation. Experiments are underway to determine global patterns of gene expression in normal and bgn deficient osteoblasts treated with or without BMP using microarrays. It is anticipated that with extensive bioinformatic analysis, we will gain additional insight about the complex circuitry that connects bgn to BMP activation and bone cell function. To date, most of our analysis of the bgn KO mice has been restricted to long bones. In order to determine what role bgn and other related SLRPs might play in craniofacial tissues, the teeth of normal and bgn or dcn (the related SLRP, decorin) KO mice were examined extensively by histology and electron microscopy. Each deficiency resulted in specific disruptions of dentin and enamel formation. Bgn and dcn deficiencies had the same overall effect on dentin formation resulting in a hypomineralized dentin, although the effect was more dramatic in the absence of dcn. In contrast, bgn and dcn deficiencies had opposite effects on enamel formation. Enamel formation was dramatically increased in bgn deficient mice whereas it was delayed in the absence of dcn. The increased enamel formation in absence of bgn directly resulted from an upregulation of amelogenin synthesis whereas delayed enamel formation in the absence of dcn was most probably an indirect consequence of the disruption in dentin formation, dentin being so porous that it prevented enamel deposition. In conclusion, our results indicate that bgn and dcn control tooth formation. The phenotypes of bgn and dcn deficient mice indicate that during tooth development, bgn represses amelogenin expression and enamel formation whereas dcn promotes dentin mineralization. Currently we are examining mice deficient in both dcn and bgn (double KO) before birth. This is being done to determine whether SLRPs play a role in controlling the structure and formation of other parts of the craniofacial complex during embryogenesis.